Microbiological process for removing non-ionic surface active agents, detergents and the like from wastewater and microorganism capable of same

ABSTRACT

A process for degrading and removing anionic and/or non-ionic surface active agents, detergents and the like from wastewater containing the same comprising treating wastewater containing the anionic and/or non-ionic surface active agents, detergents and like compounds with a novel microbial strain of Pseudomonas fluorescens under aerobic conditions; and the novel microorganism of the strain Pseudomonas fluorescens.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 158,758,filed June 12, 1980, now U.S. Pat. No. 4,317,885, which in turn is adivision of U.S. patent application Ser. No. 35,045, filed May 1, 1979,now U.S. Pat. No. 4,274,954.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a process for degrading anionic and/ornon-ionic surface active agents, detergents and like materials fromwastewater containing the same and, more specifically, to a process fortreating waste water containing anionic and/or non-ionic surface activeagents, detergents and like materials, for example, effluent fromindustrial plants, including those from textile manufacturers orprocessors, with a novel microorganism of the strain Pseudomonasfluorescens whereby the anionic and/or non-ionic surface active agents,detergents and similar materials in the wastewater are thereby degradedand removed and the wastewater is purified. Further, this inventionrelates to a novel microbial strain of the genus Pseudomonas.

2. Description of the Prior Art

Wastewaters containing organic and inorganic materials as pollutants areunsuitable for reuse and undersirable for release into the biosphere dueto problems of pollution which results when they are dischargeduntreated. To remove, or at least minimize, this difficulty, domestic,municipal and industrial wastewaters are conventionally processed inbiological treatment systems, for example, aerated lagoons or activatedsludge systems, for removal of biodegradable organic matter prior tore-use or discharge to receiving bodies of water.

While the biological processes occurring during such a biologicaltreatment provide the ability to produce effluent with lower biochemicaloxygen demand (BOD) and low chemical oxygen demand (COD), unfortunately,removal of materials such as anionic and non-ionic surface activeagents, detergents and like materials using conventionally employedbiological treatment systems has not met with a large amount of success.Even when the biological treatment system is capable of degradingmaterials such as anionic and non-ionic surface active agents,detergents and like materials, the degradation process is often too slowor insufficient resulting in a concentration build-up of these materialsor a carry through the system of these materials undigested. Reductionin the levels of anionic and non-ionic surface active agents, detergentsand similar materials in wastewater to an acceptable level for dischargeof the wastewater into the biosphere is either costly or ineffectiveparticularly for difficultly biodegradable or non-biodegradablematerials.

The difficulty arising due to the inability of bacteria normally presentin conventional biological treatment systems to degrade anionic andnon-ionic surface active agents, detergents and like materials at anacceptable rate, if at all, or in reducing the concentration thereof tothe extent that such wastewater after treatment can be released into thebiosphere has created a problem in the past. This is particularly truewhere insufficiently treated water containing these materials isreleased in areas where the water supply is naturally confined or inareas where the water table is particularly high. Numerous federal andstate regulations relating to effluent wastewater quality as to surfaceactive agents detergents and like materials concentrations have beenpromulgated and implemented to protect the biosphere. In the pastdecade, numerous changes in the synthetic detergent industry haveoccurred as a result of this problem with a shift from non-biodegradablesurface active agents and detergents to more biodegradable surfaceactive agents and detergents. Although there has been this change todomestic and industrial use of more biodegradable surface active agentsand detergents, concern still exists with the treatment of wastewaterscontaining such.

Even with the shift to more biodegradable synthetic detergents andsurface active agents, a problem still exists in the treatment ofindustrial wastewaters where, due to performance characteristics,synthetic surface active agents and detergents which would basically, inthe present state of the art, be considered to be non-biodegradable arestill employed resulting in difficulties in wastewater treatment ofeffluent from these industrial processes utilizing such. In particular,the use of microbiological treatment of wastewater effluents fromtextile plants employing non-biodegradable synthetic surface activeagents and detergents as processing aids and agents has resulted in highcost treatment of such wastewater. Further, the presence of thesesynthetic surface active agents and detergents in wastewater effluenthas caused processing difficulties due to foaming which inherentlyoccurs with increased concentration of such materials in the wastewatereffluent. This foaming during treatment of such wastewaters has thusresulted in the need for increased capital outlay to provide largertreatment facilities than would normally be required were the foamingnot to exist or has resulted in decreased through-put where capacity ofthe biological treatment facility is not increased.

With the increasing concern as to minimization of the problems arisingfrom pollution, biological processes utilizing microorganisms are beingindustrially, municipally and domestically employed and in increasngamount. In the processing of wastewaters a large amount of activity inresearch and development has occurred and is presently occurring todevelop improved microbial strains capable of use in industrial,municipal and domestic wastewater treatment facilities. While advanceshave been made in curtailing pollution problems arising from syntheticsurface active agents and detergents due to the shift to morebiodegradable materials, a sufficiently acceptable solution to theproblem of removing difficulty biodegradable or substantiallynon-biodegradable synthetic surface active agents and detergents fromwastewaters from domestic, municipal and industrial sources has not yetbeen developed.

As far as surface active agents are concerned, ethoxylated alkylphenolics and naphtholics have advantageous properties as surface activeagents and detergents and are useful industrially. Unfortunately, suchphenolics and naphtholics, particularly those in which the alkyl moietyis branched chain, are considered in the present state of the art to benon-biodegradable or difficultly biodegradable. As a result, it isnecessary to monitor wastewaters from processes in which they are usedto ensure that their levels in discharge waters meet municipal, stateand federal standards to prevent water pollution.

In view of the industrial utility of these phenolics and naphtholics andthe requirements of meeting water pollution regulations on the maximumlevels they can be present in discharged waters, an analytical methodfor determining the levels of such in water is disclosed inEnvironmental Science and Technology, 11, No. 13, p. 1167-1171 (December1977). The levels of such a nonylphenol ethylene oxide condensate having10 moles of ethylene oxide per molecule appearing in wastewater whichhad been biologically treated using semicontinuous activated sludge orcontinuous activated sludge were determined. This is the only known workwhere an alkyl phenolic or naphtholic ethylene oxide condensate has beensubjected to microbiological treatment with a reduction in the amountthereof being observed. Even here, this biological treatmentinsufficiently reduced the level of such a phenolic detergent inwastewater and the time required to achieve such was undesirably long.

SUMMARY OF THE INVENTION

Accordingly, an object of this invention is to provide a process wherebyanionic and non-ionic surface active agents, detergents, and likematerials present in domestic, municipal and industrial wastewaters canbe removed.

Another object of this invention is to provide a biological process fortreatment of industrial, municipal and domestic wastewaters to not onlyremove biodegradable organic matter therefrom but to specifically reducethe level of or remove anionic and non-ionic surface active agents,detergents and like materials therefrom.

A further object of this invention is to provide a biological treatmentprocess for removal of difficulty biodegradable or substantiallynon-biodegradable anionic and/or non-ionic surface active agents,detergents and like materials from industrial, municipal and domesticwastewaters.

Also an object of this invention is to provide a biological treatmentprocess for removal of anionic and/or non-ionic surface active agents,detergents and like materials from industrial, municipal and domesticwastewaters using a novel mutant of Pseudomonas fluorescens.

An even further object of this invention is to provide a biologicaltreatment process for industrial, municipal and domestic wastewater toremove anionic and/or non-ionic surface active agents, detergents andlike materials therefrom and render such suitable for discharge into thebiosphere, thereby minimizing problems of pollution.

An additional object of this invention is to provide a microbiologicaltreatment process capable of degrading anionic and/or non-ionic surfaceactive agents, detergents and like materials, such as the ethylene oxidecondensates of alkyl phenolics and naphtholics, which have beenpreviously thought to be substantially non-biodegradable or extremelydifficult to biodegrade.

A further object of this invention is to provide a treatment forindustrial, municipal and domestic wastewaters utilizing a novel mutantof Pseudomonas fluorescens, alone or in combination with othermicroorganisms, to degrade and remove synthetic anionic and/or non-ionicsurface active agents, detergents and like materials from wastewaters.

An additional object of this invention is to provide a novel mutantstrain of the species Pseudomonas fluorescens.

In one embodiment of this invention, this invention provides a processfor treating wastewater containing anionic and/or non-ionic surfaceactive agents, detergents and like materials, which comprises treatingwastewater containing synthetic non-ionic surface active agents,detergents and like materials with a microorganism of the strainPseudomonas fluorescens 3P.

In another embodiment of this invention, this invention provides a novelstrain of the species Pseudomonas fluorescens 3P having thecharacteristics described below.

DETAILED DESCRIPTION OF THE INVENTION

The novel mutant Pseudomonas fluorescens 3P (hereinafter "mutantstrain") was produced by selection and subjecting to mutation techniquesof strains of microorganisms isolated from the soil and wastewater froma wastewater lagoon at a large textile chemical manufacturing plantlocated in Wellford, S.C.

This novel mutant strain has been found to be capable of degrading andremoving anionic and/or non-ionic surface active agents, detergents andlike materials, previously thought to be difficultly biodegradable orsubstantially non-biodegradable and has the characteristics set forthbelow.

The mutant strain Pseudomonas fluorescens 3P is a gram-negative,non-spore-forming rod. The cells are straight rods which have asingle-polar flagellum, and the cells are motile. In culture,approximately 1% of the cells exist in the form of long filaments ofgreater than five cell units long. On Kings Medium A (described in E. O.King et al., J. Lab. & Clin. Med., Volume 44, No. 2, page 303 (1954),and on Difco BACTO-Antibiotic Medium 3 (trade name produced by DifcoLaboratories), solidified with agar at temperatures from 20°-30° C., afluorescent yellow diffusible pigment is formed. No noticeable odors aregiven off by cultures of Pseudomonas fluorescens 3P on complex mediasuch as nutrient broth and nutrient agar, or minimal salts-based mediacontaining a carbon source such as glucose.

The mutant strain Pseudomonas fluorescens 3P is capable of growing on aglucose containing minimal salts medium (Roy Curtiss, III, J. Bact., 89,pages 28-40 (1965)) containing ammonium ion as a nitrogen source, thusdemonstrating the strain does not appear to require any growth factor orvitamin supplement. The mutant strain does not utilize acetate as a solecarbon source.

The mutant strain is an obligate aerobe. Growth is not possibleanaerobically in the presence of nitrate and the mutant strain does notproduce nitrate reductase. The cells of the mutant strain are incapableof accumulating poly-β-hydroxybutyric acid granules even thoughDL-hydroxbutyrate serves as a sole carbon source.

A suitable growth temperature range is about 20°-37° C., with optimalgrowth occurring at 30° C. No growth is observed in ten days at 14° C.The strain displays arginine dihydrolase activity and is incapable ofgelatin hydrolysis.

Other cultural characteristics and colonial morphology of this mutantstrain are shown in Tables 1-6 below.

In the following tables, Pseudomonas fluorescens (ATCC 13525) wasemployed as a known type strain for characterization purposes.

                  TABLE 1                                                         ______________________________________                                        MICROSCOPIC MORPHOLOGY                                                                      STRAIN                                                                          PSEUDOMONAS                                                   CHARACTERISTIC  FLUORESCENS** 3P                                              ______________________________________                                        Cell Size*                                                                    Length          2.3-2.8       2.3-2.8                                         Width           0.7-0.8       0.7-0.8                                         Gram Reaction   Negative      Negative                                                        rod           rod                                             ______________________________________                                         *Wet mounts of tenhour cultures (late exponential phase) viewed under         phase contrast (1000×). Sizes given in micrometers.                     **Data from Bergey's Manual of Determinative Bacteriology, 8th Ed., The       Williams & Wilkins Co., Baltimore (1974).                                

                  TABLE 2                                                         ______________________________________                                        COLONIAL CHARACTERISTICS OF                                                   PSEUDOMONAS FLUORESCENS 3P                                                    After 48 Hours At 35° C.                                               ______________________________________                                        Plate Count Agar                                                              Slightly irregular colonies are umbonate with a rough                         surface. They are opaque, four to five mm in diameter and                     have a slightly undulate edge. They are white in color                        and no pigments are produced in forty-eight hours.                            Nutrient Agar                                                                 Circular, convex colonies have a wrinkled surface.                            They are opaque, three to five mm in diameter and have an                     undulate edge. Colonies are white and no pigments are                         produced in forty-eight hours.                                                Hektoen Enteric Agar                                                          Colonies are circular, convex and have a smooth                               surface. They are opaque, green, two to three mm in                           diameter and have an entire edge. No pigments are                             produced.                                                                     Pseudosel Agar                                                                Colonies are irregular, raised, have a smooth surface                         and an undulate edge. They are one to two mm in diameter,                     opaque and white in color. A diffusible fluorescent                           pigment is produced.                                                          T-soy Agar                                                                    Circular, slightly convex colonies have a smooth                              surface and an entire edge. They are two to four mm in                        diameter, opaque and white in color. A yellow-green                           diffusible fluorescent pigment is produced.                                   ______________________________________                                          NOTE:                                                                        Plate Count Agar and Hektoen Enteric Agar are products of Difco               Laboratories. Pseudosel Agar, Nutrient Agar and Trypticase Soy Agar are       products of Baltimore Biological Laboratories.                           

                  TABLE 3                                                         ______________________________________                                        UTILIZATION OF CARBON-                                                        CONTAINING COMPOUNDS FOR GROWTH                                                              GROWTH RESPONSE**                                                               PSEUDOMONAS                                                  COMPOUND*        FLUORESCENS  3P                                              ______________________________________                                        Carbohydrates (&                                                              Sugar Derivatives)                                                            α-Cellulose                                                                              -            +                                               L-Arabinose                   -                                               D-Ribose         -            -                                               D-Glucose                     +                                               Sucrose                       -                                               Trehalose                     -                                               D-Cellulose      -            +                                               Xylose           -            -                                               Organic Acids                                                                 Acetate                       -                                               Propionate       -            -                                               Bytyrate                      +                                               Isobutyrate      -            -                                               Valerate         -            -                                               Caproate         -            -                                               Heptanoate       -            -                                               Caprate          -            ±                                            Stearate         -            +                                               Dicarboxylic Acids                                                            Maleate          -            +                                               Malonate         -            -                                               Succinate        -            -                                               Glutarate        -            -                                               Saccharate                    +                                               Hydroxyacids                                                                  L-Malate         -            -                                               DLβ-Hydroxybutyrate      +                                               DL-Lactate       -            -                                               DL-Glycerate     +            +                                               Miscellaneous                                                                 Organic Acids                                                                 Citrate          -            -                                               α-Ketoglutarate                                                                          -            -                                               Pyruvate         -            +                                               Polyhydric Alcohols                                                           and Glycols                                                                   Mannitol         -            -                                               Glycerol         -            +                                               Propyleneglycol               +                                               m-Inositol                    -                                               Sorbitol                      -                                               Alcohols                                                                      Ethanol                       +                                               n-Propanol       -            +                                               n-Butanol        -            +                                               Non-Nitrogenous                                                               Aromatic and Other                                                            Cylic Compounds                                                               Benzoate         -            -                                               Aliphatic Amino Acids                                                         Lα-Alanine -            +                                               Dα-Alanine -            +                                               β-Alanine                +                                               L-Leucine        -            +                                               L-Aspartate      -            +                                               L-Glutamate      +            +                                               L-Lysine         -            +                                               DL-Arginine                   +                                               L-Valine                      ±                                            Glycine                       -                                               Asparagine                    +                                               Amino Acids and                                                               Related Compounds                                                             Containing A                                                                  Ring Structure                                                                L-Histidine      -            +                                               L-Proline        -            +                                               L-Tyrosine       -            -                                               Miscellaneous                                                                 Nitrogenous Compounds                                                         Betaine          -            +                                               Sarcosine        -            -                                               Acetamide        -            +                                               Glucosamine      -            -                                               Detergents***                                                                 Igepal CO 520    -            +                                               (2000 mg/l)                                                                   Igepal CO 610    -            +                                               (2000 mg/l)                                                                   Igepal CO 660    -            +                                               (2000 mg/l)                                                                   ______________________________________                                         *Compound added at 0.5% to minimal salts medium (Curtiss (1965)).             **+ indicates growth greater than blank;   indicates growth less than tha     of blank; ± indicates growth approximately equal to blank or weak          growth, after seven days at 30° C.                                     ***Trade name for a nonionic nonyl phenolethylene oxide condensate            produced by GAF.                                                         

                  TABLE 4                                                         ______________________________________                                        UTILIZATION OF NITROGENOUS                                                    COMPOUNDS AS SOLE NITROGEN SOURCE                                                          GROWTH RESPONSE**                                                               PSEUDOMONAS                                                    COMPOUND*      FLUORESCENS  3P                                                ______________________________________                                        NH.sub.4 Cl    -            +                                                 KNO.sub.3      -            +                                                 L-Glutamate    -            +                                                 L-Aspartate    -            ±                                              L-Alanine      -            +                                                 ______________________________________                                         *Compound added at 0.5 g/100 ml to minimal salts medium (Curtiss (1975)       but without NH.sub.4 Cl and NH.sub.4 NO.sub.3) consisting of 0.5 g of         Dglucose/100 ml.                                                              **+ indicates growth greater than blank;   indicates growth less than tha     of blank; ± indicates growth approximately equal to blank or weak          growth, after seven days at 30° C.                                

                  TABLE 5                                                         ______________________________________                                        CULTURE GROWTH IN PRESENCE OF HEAVY METALS                                                     STRAIN RESPONSE**                                            HEAVY                  PSEUDOMONAS                                            METAL*  CONCENTRATION  FLOURESCENS  3P                                        ______________________________________                                        HgSO.sub.4                                                                            2 × 10.sup.-3 M                                                                        -            +                                                 10.sup.-3 M    -            -                                                 10.sup.-4 M    -            +                                                 10.sup.-5 M    -            +                                         CdCl.sub.2                                                                            2 × 10.sup.-3 M                                                                        -            -                                                 10.sup.-3 M    -            -                                                 10.sup.-4 M    -            +                                                 10.sup.-5 M    -            +                                         CoCl.sub.2                                                                            2 × 10.sup.-3 M                                                                        -            -                                                 10.sup.-3 M    -            -                                                 10.sup.-4 M    -            +                                                 10.sup.-5 M    +            +                                         AgSO.sub.4                                                                            2 × 10.sup.-3 M                                                                        -            -                                                 10.sup.-3 M    -            -                                                 10.sup.-4 M    -            -                                                 10.sup.-5 M    -            -                                         Na.sub.2 HAsO.sub.4                                                                   2 × 10.sup.-3 M                                                                        -            +                                                 10.sup.-3 M    -            +                                                 10.sup.-4 M    -            +                                                 10.sup.-5 M    -            +                                         ______________________________________                                         *Heavy metal added to minimal salts medium containing (0.5%) Dglucose         (Curtiss (1965)).                                                             **Growth response scored as: + indicates growth (no inhibition);              indicates no growth (inhibition).                                        

                  TABLE 6                                                         ______________________________________                                        RESISTANCE TO ANTIBIOTICS                                                                  STRAIN GROWTH RESPONSE                                           ANTIBIOTIC   3P                                                               ______________________________________                                        Ampicillin    R*                                                              Carbenicillin                                                                              R                                                                Cephalothin  R                                                                Chloramphenicol                                                                            R                                                                Coly-mycin   S                                                                Gentamicin   S                                                                Kenamycin    S                                                                Mandol       R                                                                Streptomycin I                                                                Tobramycin   S                                                                Tetracycline R                                                                Amikacin     S                                                                ______________________________________                                         *Growth response on Pfizer Antimicrobial Susceptibility Disks; Pfizer,        Inc. scored: S = sensitive to antibiotic; R = resistant to antibiotic; I      intermediate.                                                            

On the basis of the morphological, cultural and physiologicalcharacteristics set forth above, the strain has been identified as amember of the species, Pseudomonas fluorescens and has been designatedherein as Pseudomonas fluorescens 3P. A culture of the strain has beendeposited in the American Type Culture Collection and has received anaccession number, ATCC-31483.

The mutant strain as described above was selected and subjected tomutagenesis techniques in the following manner.

A sample of the soil and wastewater from a large producer of industrialchemicals for the textiles industry was obtained from a lagoon used forholding wastewater effluent from the plant. Several types ofmicroorganisms were isolated from the water sample using an agar mediumwith 1% by weight of an ethoxylated nonylphenol detergent having 10moles on the average of ethylene oxide per mole of nonylphenol addedthereto (Igepal CO 660) as the only source of carbon. The composition ofthe media used is set forth in Table 7 below.

                  TABLE 7                                                         ______________________________________                                        COMPOSITION OF SOLE SOURCE OF CARBON AGAR                                     Compound               Quantity                                               ______________________________________                                        (NH.sub.4).sub.2 SO.sub.4                                                                            1.0    g/l                                             MgSO.sub.4.7H.sub.2 O  0.2    g/l                                             KH.sub.2 PO.sub.4      1.5    g/l                                             CaCl.sub.2             10     mg/l                                            FeSO.sub.4.7H.sub.2 O  5.0    mg/l                                            K.sub.2 HPO.sub.4      3.5    mg/l                                            MnSO.sub.4             2.0    ml/l                                            Trace Element Solution 1.0    ml/l                                            H.sub.3 BO.sub.3       10     mg/l                                            Li.sub.2 SO.sub.4      70     mg/l                                            MoO.sub.3              10     mg/l                                            Ethoxylated Nonyl-Phenol %                                                    Detergent (as carbon source)                                                                         1%                                                     Agar                   15     g/l                                             pH adjusted to         7.2                                                    ______________________________________                                    

Subsequently, to eliminate carryover of nutrient, a second transfer ofthe microorganisms to agar containing 0.1% by weight of the ethoxylatednonylphenol detergent as described above was conducted.

As a result of the above procedures, eight microorganism culturesshowing the heaviest growth were then grown in a liquid medium of thecomposition shown in Table 7 above (but without the agar). Each of theseeight cultures of microorganisms was then subjected to a mutagenesisusing 0.02% sodium nitrite at a pH of 6.5-6.8 as described by J. H.Miller, Experiments in Molecular Genetics, Cold Spring HarborLaboratory, New York (1972), in order to stimulate enzymatic changes inthe microorganism cultures.

Of the cultures which were treated as described above, those which grewbetter than the original cultures were then re-inoculated onto an agarmedium containing 0.1% by weight ethoxylated nonylphenol detergent asthe only source of carbon and then maintained in test tubes containingan aqueous solution of the ethoxylated nonyl phenol detergent in aconcentration of 200 mg/l of the ethoxylated nonyl phenol detergent.

Additional cultures were developed by using non-sterile soilpercolators. The soil percolators consisted of a side arm suction flaskconnected to a soil chamber by means of a draft tube through which 50%v/v wastewater fortified with 1.0 g/l (NH₄)SO₄ and 3.5 mg/l disodiumphosphate and 200 ppm of the ethoxylated nonylphenol was drawn up andallowed to trickle down through the soil media containing the mutatedorganisms. By this process organisms were further selected and adaptedto survive in field conditions. Approximately 75% of the liquid mixturewas periodically replaced by fresh solution and this procedure wascontinued for a six-week period.

At the end of this period of time, three organisms which predominatedwere isolated from the soil percolators.

All of the microorganism isolates described above were tested for growthon an agar medium containing 200 mg/l of the ethoxylated nonylphenoldetergent as the sole source of carbon, on an agar medium containing 200mg/l of the ethoxylated nonylphenol detergent plus 20% by weight of thewastewater taken from the lagoon of the textile chemical producing plantand tubes of 20% by weight of such wastewater with 200 mg/l of theethoxylated nonylphenol detergent were added. All of the cultures showedgrowth in these media.

In order to efficiently remove detergent materials, in 100% industrialwastewater containing such, the cultures obtained as described abovewere first acclimated to a 40% concentration of such industrialwastewater and then were placed in 100% of such wastewater.

To determine effectiveness in terms of the activity of these cultures,an analysis for nonionics as described by D. G. Stevenson, "TheAdsorptiometric Determination of A Non-Ionic Detergent", Dept. of AtomicEnergy, Atomic Weapons Research Establishment, Aldermaston, Berkshire,England (August, 1954), was employed.

All of the cultures treated as described above proved to be capable oftolerating 100% of the industrial wastewater. These microorganismcultures were then each inoculated into a 200 mg/l solution of theethoxylated nonylphenol detergent plus inorganic salts, shown in Table 7above, in the concentrations set forth in Table 7 (hereinafter"detergent-salts") for further selection as to biodegradation ability.

The three most effective cultures of microorganisms obtained asdescribed above were then inoculated into solutions of 200 mg/l of the"detergent-salts" placed in shake flasks along with a control, and allof the flasks were shaken for a week. The solution contained in eachflask was then analyzed for degradation of the detergent using aninfrared method of analysis for oil and grease as disclosed in StandardMethods, 14th Ed. "Partition-Infrared Method for Grease and Oil", pp.516-517, American Public Health Association, Washington, D.C. (1975).

The most effective culture for biodegradation of the synthetic detergentdescribed above was found to be a novel strain of the speciesPseudomonas fluorescens and is designated herein as Pseudomonasfluorescens 3P.

The Pseudomonas fluorescens 3P can be employed alone or in combinationwith other microorganisms conventionally used in microbiologicaltreatment of wastes. This invention also includes the use of anyvariants of Pseudomonas fluorescens 3P alone or in combination.

The mutant strain Pseudomonas fluorescens 3P used in this invention canbe cultured in wastewater from any type of industrial plant containinganionic and/or nonionic surface active agents, detergents and likematerials either using a batch process, a semi-continuous process or acontinuous process, and such is cultured for a time sufficient todegrade the anionic and/or nonionic surface active agents, detergentsand like materials present in the wastewater and remove them or breakthem down into components capable of being degraded by other organismsnormally found in biological wastewater treatment systems.

The mutant strain of this invention can be employed in ion exchangeresin treatment systems, in tricking filter systems, in carbonadsorption systems, in activated sludge treatment systems, in outdoorlagoons or pools, etc. Basically, all that is necessary is for themicroorganism to be placed in a situation of contact with the wastewatereffluent. In order to degrade the material present in the wastewater,the organisms can be cultured under conditions of about 15° C. to about42° C., preferably about 20° C. to about 38° C. Desirably, the pH ismaintained in a range of about 5.5 to about 8.5, preferably 6.5 to 8.0Control of the pH can be by monitoring of the system and an addition ofappropriate pH adjusting materials to achieve this pH range.

The culturing is conducted basically under aerobic conditions of adissolved oxygen concentration of about 2 ppm or more, preferably about5 ppm or more. These conditions can be simply achieved in any mannerconventional in the art and appropriate in the treatment system designbeing employed. For example, air can be bubbled into the system, thesystem can be agitated, a trickling system can be employed, etc.

The wastewater to be subjected to the process of this invention maycontain sufficient nitrogen and phosphorus for culturing without theneed for any additional source of nitrogen or phosphorus being added.However, in the event the wastewater is deficient in these twocomponents, suitable available nitrogen sources, such as ammonia or anammonium salt, e.g., ammonium sulphate, can be added to achieve anavailable nitrogen content of at least about 10 ppm or more per 100BOD₅. Similarly, phosphorus can be supplemented, if necessary, byaddition of orthophosphates, e.g., sodium phosphate, to achieve aphorphorus level in the wastewater of about 1 ppm or more per 100 BOD₅.In general, the treatment is conducted for a sufficient time to achievethe reduction in levels of nonionic surface active agents, detergentsand like materials and, in general, about 24 hours to eight weeks orlonger, although this will depend upon the temperature of culturing, theconcentration of these materials in the wastewater and the volume to betreated and other factors, has been found to be suitable.

In the above manner, anionic and/or nonionic surface active agents,detergents and like materials which have been previously considered inthe art to be difficultly degradable or non-biodegradable, as well asother organic compounds which might be present in wastewater systems,can be advantageously treated to provide treated wastewater suitable fordischarge after any additional conventional processing such as settling,chlorination, etc., into rivers and streams.

As can be seen from an examination of the examples given hereinbelow,the mutant strain Pseudomonas fluorescens 3P provides advantageousresults in degrading synthetic anionic and non-ionic surface activeagents, detergents and the like.

The following description is not to be considered to be limiting, rathermerely exemplary of the types of anionic and non-ionic surface activeagents, detergents and like materials to which this invention isapplicable and which can be degraded in accordance with the method ofthis invention.

Examples of non-ionic materials include non-ionic alkanolamides;ethoxylated aliphatic alcohols such as ethoxylated lauryl alcohol,ethoxylated coco alcohol, ethoxylated mysteryl alcohol, etc.;ethoxylated aklylphenols and naphthols, such as ethoxylatednonylphenols, ethoxylated dodecylphenol, ethoxylated tetradecylphenol,etc.; polyalkylene oxides such as polyethyleneoxide condensates ofvarying molecular weights; ethoxylated fatty acids such as ethoxylatedstearates, laurates and palmitates, etc.; ethoxylated glycol esters andglycerol esters such as ethoxylated glycosides and glycerides; andsimilar materials.

Examples of anionic materials include anionic alkyl sulfates such aslauryl sulfate, palmityl sulfate, etc.; alkyl sulfonates such as laurylsulfonate, etc.; alkylethyleneoxy sulfonates, such as coco alcoholethyleneoxy sulfonates; sulfosuccinates such as dioctylsulfosuccinate,etc.; alkylbenzene and naphthylene sulfonates such as tetradecylbenzenesulfonate, etc., and like materials.

Most of these types of anionic and non-ionic surface active agents anddetergents are well known and are commercially available. Representativeexamples include Consowet (a dioctylsulfosuccinate anionic detergentproduced by Consos, Inc., Charlotte, N.C.), Astrowet (adioctylsulfosuccinate anionic detergent produced by Astro AmericanChemical Co., Greenville, S.C.) and Merpol (a non-ionic ethylene oxidecondensate produced by E. I. du Pont de Nemours and Co., Inc.) Ingeneral, the basic chemical structure or nature of these materials isnot limiting as long as they can be considered to be anionic or nonionicsurface active agents or detergents.

In order to further demonstrate the effectiveness of the strain ofPseudomonas fluorescens 3P, the following examples are given asexemplary of the invention but without intending to limit the same.Unless otherwise indicated herein, all parts, percents, ratios and thelike are by weight.

EXAMPLE 1

A biotower which was a trickling filter was used. The biotower compriseda reservoir for a liquid and a column containing Pall rings of a plasticresin with one end of the column being placed just above the liquid inthe reservoir. A pump was submerged in the liquid reservoir forrecycling liquid from the liquid reservoir through a tube to the top ofthe column for dispersion of the liquid down through the initial Pallring packing. A synthetic liquid feed comprising 2% by weight sweet wheyand 200 mg/l disodium phosphate and inoculated with Pseudomonasfluorescens 3P was used and circulated through the biotower in themanner described above until a slime layer of microrganisms formed. Asolution of 520 mg/l detergent-salts was fed thereinto on a batch basis.The liquid was then circulated through the biotower for a period of 41.5hours and samples were removed periodically and analyzed for ethoxylatednonylphenol having 10 moles of ethylene oxide per mole of nonylphenol.

The results showed that from an initial ethoxylated nonylphenoldetergent concentration of 520 mg/l, such concentration was reduced to65 mg/l after 41.5 hours, a degree of removal of 88%.

EXAMPLE 2

A sample of a culture of the mutant strain Pseudomonas fluorescens 3P,along with a control, was inoculated into a shake flask containing 200mg/l of the "detergent-salts" and the shake flask containing themicroorganism and the control shake flask were each shaken for one week.

Thereafter, the liquid in each shake flask was analyzed for ethoxylatednonylphenol detergent and the following results in Table 8 wereobtained.

                  TABLE 8                                                         ______________________________________                                               Detergent Concentration                                                                       % Detergent                                            Sample   Initial     Final     Degradation                                    ______________________________________                                        3P       200         120       40                                             Control  200         200        0                                             ______________________________________                                    

EXAMPLE 3

The procedures of Example 2 were repeated using a wastewater obtainedfrom the lagoon of a textile chemical producing plant fortified with 200mg/l of ethoxylated nonylphenol were repeated, again also using acontrol.

After one week, the ethoxylated nonylphenol concentration had beenreduced to 143 mg/l whereas in the control, the ethoxylated nonylphenoldetergent concentration was 193 mg/l, substantially the same as that ofthe initial concentration of ethoxylated nonylphenol. Thus, thePseudomonas fluorescens 3P microorganism accomplished a biodegradationof 25% more of the ethoxylated nonylphenol detergent than that achievedin the control.

EXAMPLE 4

Experimentation was performed to demonstrate the effectiveness ofdegradation of Tide (trademark for a household detergent containing ananionic detergent produced by the Proctor and Gamble Co.) by Pseudomonasfluorescens 3P.

Two-hundred-fifty ml shake flasks containing 100 mg of Tide/100 mlnutrient broth were used. After seven days, Pseudomonas fluorescens 3Phad degraded approximately fifty percent of the Tide present. Anotherflask as a control inoculated simply with a microorganism of a Bacillusspecies showed poor, if any growth.

Analysis was performed by the methylene blue active substances method(14th Edition, Standard Methods for the Examination of Water andWastewater, 1975), with the exception that the standard curve in themethod was made using Tide in place of the linear alkylate sulfonate.

While the invention has been described in detail and with respect tospecific embodiments thereof, it will be apparent to one skilled in theart that changes and modifications can be made therein without departingfrom the spirit and scope thereof.

What is claimed is:
 1. A microorganism Pseudomonas fluorescens 3P havingthe identifying characteristics of ATCC-31483, said microorganism beingcapable upon culturing in wastewater containing anionic and/or nonionicsurface active agents, and detergents of utilizing such as anassimilable source of carbon.